Project Summary Lipids control many critical biological processes, and aberrant lipid production and localization lead to the onset of many diseases. In spite of this significance, many critical details of lipid biosynthesis and trafficking remain poorly understood due to longstanding impediments to tracking lipid biosynthesis and subcellular localization. Herein, we present a comprehensive plan for overcoming these challenges by developing synthetic probes that act as tagged analogs of biosynthetic precursors of lipids, thus enabling the detection and imaging of derivatized products in their native environment and in real time. Towards this end, we are first designing and synthesizing analogs of lipid precursors containing small tags that can be modified using click chemistry to achieve the selective labeling of lipid products within cells. In this endeavor, a number of metabolite analog designs are being pursued to optimize labeling of lipid products and to access compounds that label different lipid products depending on their specific structures. Following synthesis, each compound will be rigorously evaluated to assess incorporation into different potential products and to optimize infiltration of biosynthetic pathways. A range of tools will be exploited and developed for this purpose, including mass spectrometry-based tracking of lipid products in biological samples, TLC analysis to confirm the families of lipids that are labeled, in vitro assays to probe the ability of synthetic compounds to act as substrates for enzymatic modification, and fluorescence microscopy to confirm the production of lipid products and characterize product localization. Once each of these analytical approaches has been optimized, these platforms will be implemented to answer biological questions pertaining to the localization of lipids during biosynthesis and trafficking events. These studies will be accomplished by applying the proposed mass spectrometry and fluorescence microscopy methods in conjunction with a series of mutant cell lines with modified lipid biosynthetic machinery as well as cellular fractionation experiments.